Information regarding the structure and function of living tissues and cells is instrumental to the advancement of cell biology and biophysics. Nonlinear optical microscopy can provide such information, but only certain biological structures generate nonlinear optical signals. Therefore, structural specificity can be achieved by introducing labels for nonlinear optical microscopy. Few studies exist in the literature about labels that facilitate harmonic generation, coined "harmonophores". This thesis consists of the first major investigation of harmonophores for third harmonic generation (THG) microscopy. Carotenoids and chlorophylls were investigated as potential harmonophores. Their nonlinear optical properties were studied by the THG ratio technique. In addition, a tunable refractometer was built in order to determine their second hyperpolarizability (γ). At 830 nm excitation wavelength, carotenoids and chlorophylls were found to have large negative γ values however, at 1028 nm, the sign of γ reversed for carotenoids and remained negative for chlorophylls. Consequently, at 1028 nm wavelength, THG signal is canceled with mixtures of carotenoids and chlorophylls. Furthermore, when such molecules are covalently bonded as dyads or interact within photosynthetic pigment-protein complexes, it is found that additive effects with the γ values still play a role, however, the overall γ value is also influenced by the intra-pigment and inter-pigment interaction.
The nonlinear optical properties of aggregates containing chlorophylls and carotenoids were the target of subsequent investigations. Carotenoid aggregates were imaged with polarization-dependent second harmonic generation and THG microscopy. Both techniques revealed crystallographic information pertaining to H and J aggregates and β-carotene crystalline aggregates found in orange carrot. In order to demonstrate THG enhancement due to labeling, cultured cells were labeled with carotenoid incorporated liposomes. In addition, Drosophila melanogaster larvae muscle as well as keratin structures in the hair cortex were labeled with β-carotene.
Polarization-dependent THG studies may be particularly useful in understanding the structural organization that occurs within biological structures containing carotenoids and chlorophylls such as photosynthetic pigment-protein complexes and carotenoid aggregates in plants and alga. Further, artificial labeling with carotenoids and chlorophylls may be useful in clinical applications since they are nontoxic, nutritionally valuable, and they can aid in visualizing structural changes in cellular components.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/65759 |
| Date | 01 September 2014 |
| Creators | Tokarz, Danielle Barbara |
| Contributors | Barzda, Virginijus, Fekl, Ulrich |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0022 seconds